Ri-mediated Transformation of <Emphasis Type="Italic">Glycyrrhiza uralensis</Emphasis> with a Squalene Synthase Gene (GuSQS1) for Production of Glycyrrhizin

Root of Glycyrrhiza uralensis, one of the most important medicinal plants, containing bioactive triterpene saponins (glycyrrhizin). Squalene synthase (SQS) plays a regulatory role in the biosynthesis of triterpene saponins. In the present investigation, SQS coding sequence from G. uralensis was cloned by polymerase chain reaction (PCR) and a transgenic system was developed for G. uralensis through Agrobacterium rhizogenes-mediated transformation. The SQS gene placed under a CaMV 35S promoter was transferred into G. uralensis using A. rhizogenes strain ACCC10060. The transformed hairy roots were selected on Murashige and Skoog (1962)-containing phosphinothricin (PPT) and root lines were established. The integration of SQS gene was confirmed by PCR and Southern blot. Three transgenic root lines UP1, UP24, UP31 were obtained and their growth rates were detected. The result showed that transgenic root lines but UP1 line grew faster than control hairy roots; high-performance liquid chromatography (HPLC) analysis demonstrated the highest glycyrrhizin content of transgenic roots was 2.5 mg/g dry weight and was about 2.6 times higher than control hairy roots. The nucleotide sequences GuSQS1 and GUSQS2 reported in this paper appear in the EMBL nucleotide sequence database with the accession number AM182329 and AM182330, respectively.     

Effect of molybdenum on secondary metabolic process of glycyrrhizic acid in Glycyrrhiza uralensis Fisch.

A pot experiment was conducted to investigate into effects of molybdenum (Mo) on the secondary metabolic process of glycyrrhizic acid (GA). One-year-old seedlings were grown in pots with washed vermiculite and sand. Hoagland nutrition solution was irrigated with four concentrations: 0, 0.52, 5.2 and 10.4 mg L⁻¹. The accumulations of GA and its biosynthetic precursors (β-amyrin and squalene) and then expression of the key synthase (β-amyrin synthase, β-AS) were studied on 35, 70 and 105 d. In the early stage, that was on the 35 and 70 d, the contents of squalene and GA, and the expression of β-AS gene under 0.52 and 5.2 mg L⁻¹ Mo treatments were significantly higher than that under 0 and 10.4 mg L⁻¹ Mo. There was a contrary result of β-amyrin. However, the content of squalene under 0 mg L⁻¹ Mo was the highest on 105 d. Thus, it suggested an appropriate concentration of Mo could promote the accumulation of GA, by affecting the biosynthetic process of GA at a certain time. Practically, the time and amount of application of Mo on Glycyrrhiza uralensis should be the noted.     

意大利苍耳与乌拉尔甘草种间竞争能力比较

意大利苍耳已经开始入侵乌拉尔甘草农田,然而对其入侵后果目前知之甚少。通过研究意大利苍耳与乌拉尔甘草的种间竞争关系,以期为意大利苍耳对乌拉尔甘草农田生态系统的入侵能力及入侵后果的评估提供试验依据。模拟了甘草农田的土壤水肥条件,采用取代试验法,设置意大利苍耳与乌拉尔甘草的单种种植和混种种植2种种植模式,待意大利苍耳生育期结束后进行收获,分别比较了该两种植物的个体生长及生物量积累在单种种植与混种种植两种处理间的差异,并比较单种和混种模式下乌拉尔甘草的地下器官中甘草酸含量的差别,通过计算相对产量、相对竞争强度和竞争攻击系数来比较该两种植物种间竞争能力的相对强弱。结果表明：与单种模式相比,混种模式下意大利苍耳个体生长的形态学性状和有性繁殖能力均有显著增加,其株高、冠幅和种子数量较单种处理分别增加了13%、27%和56%。而乌拉尔甘草的个体生长及克隆繁殖能力均显著降低,其根瘤的形成也受到显著的抑制,混种处理的乌拉尔甘草的株高、冠幅、根总长度、根总表面积、根平均直径以及根瘤数量比单种处理分别下降了35%、45%、55%、63%、19%和76%。单种处理下每株甘草根状茎的平均条数为3条,而与意大利苍耳混种后,其根状茎的发育被完全抑制。与单种处理相比,混种处理中的意大利苍耳生物量积累均显著的增加了,其中根、茎、叶、果实及总生物量与单种模式相比分别增加了84%、73%、84%、73%和77%,而混种模式却极显著降低了乌拉尔甘草生物量和地下器官甘草酸含量的积累,使其根、茎、叶、总生物量以及甘草酸含量与单种模式相比分别下降了72%、80%、65%、71%和63%。混种模式中的意大利苍耳相对产量（RY_a）大于1,而乌拉尔甘草相对产量（RY_b）小于1,表明意大利苍耳受到邻株同种其他个体的种内竞争压力大于来自邻株乌拉尔甘草的种间竞争压力,而乌拉尔甘草受到邻株同种其他个体的种内竞争压力则小于来自邻株意大利苍耳的种间竞争压力。混种模式中的意大利苍耳相对竞争强度（RCI_a）小于0,其竞争攻击系数（A_q）大于0,而乌拉尔甘草的相对竞争强度（RCI_b）则在0-1之间,其竞争攻击系数（A_b）小于0,表明在该两种植物的混生群落中,乌拉尔甘草的竞争能力弱于意大利苍耳。总体来看,在二者混生的群落中,意大利苍耳在竞争中占据明显的优势地位,对乌拉尔甘草的产量和品质均造成强烈的负面影响。     

The constituents of licorice (Glycyrrhiza uralensis) differentially suppress nitric oxide production in interleukin-1β-treated hepatocytes

Licorice (Glycyrrhizae radix) is the roots and stolons of Glycyrrhiza uralensis Fischer or Glycyrrhiza glabra Linnaeus in the Japanese Pharmacopoeia. Glycyrrhizae radix has been widely used as a sweetener and a traditional medicine. A Glycyrrhizae radix extract contains many constituents and has antispasmodic, antitussive, anti-ulcer, and anti-inflammatory effects. However, reports comparing the anti-inflammatory effects of these constituents are very few. Here, we purified several constituents from the roots and stolons of G. uralensis and examined and compared their anti-inflammatory effects by monitoring the levels of the inflammatory mediator, nitric oxide (NO), in interleukin (IL)-1β-treated rat hepatocytes. From the G. uralensis extract, we purified the main constituent glycyrrhizin and the constituents that are characteristic of G. uralensis (chalcones and flavanones). These constituents suppressed NO production in IL-1β-treated rat hepatocytes, and isoliquiritigenin showed the greatest suppression activity. Isoliquiritigenin, isoliquiritin, and liquiritigenin significantly decreased both protein and mRNA for the inducible nitric oxide synthase. These constituents reduced the levels of mRNAs encoding tumor necrosis factor α and IL-6. In contrast, although glycyrrhizin is abundant, it showed a 100-fold lower potency in NO suppression. Therefore, both glycyrrhizin and the minor constituents (isoliquiritigenin, isoliquiritin, and liquiritigenin) may be responsible for the anti-inflammatory effects of G. uralensis. It is also implied that these constituents may have a therapeutic potential for inflammatory hepatic disorders.     

基于转录组测序对油茶角鲨烯合酶基因的克隆及分析

油茶是中国重要的木本油料树种,而角鲨烯在茶籽油中含量多少是茶油品质的重要指标,为提高油茶角鲨烯含量,以油茶种子转录组测序为基础,根据Unigene设计SQS基因RACE引物,克隆出基因全长共1 490 bp,开放阅读框1 266 bp,编码422个氨基酸,将氨基酸序列与其他植物SQS进行比对,构建进化树,分析物种间进化关系,进行蛋白质的生物信息学分析,包括蛋白质结构特点、理化性质、氨基酸组成及稳定性分析; 跨膜区域分析; 信号肽识别位点; 磷酸化位点; 二级结构及功能; 结构域分析。通过转录组测序和荧光实时定量分析了SQS基因在油茶种子发育各时期表达量变化规律,并提取各时期油脂,测定角鲨烯含量,发现两种基因表达量分析方法的结果有一致的变化趋势,且与各月份间角鲨烯含量有相同的变化规律。证明了转录组测序的有效性,并推测油茶角鲨烯合酶基因的表达量变化与角鲨烯含量的多少有直接关系,为后续从分子手段提高茶籽油中角鲨烯含量提供了理论基础。     

Development of selection method for Glycyrrhiza uralensis superior clones with high-glycyrrhizic acid contents using DNA sequence polymorphisms in glycyrrhizic acid biosynthetic genes

Glycyrrhiza plants are important resources for sweeteners and medicines, because underground parts of them contain glycyrrhizic acid (GL), which has sweet taste and various pharmacological activities (ex. anti-inflammatory, antiallergy, antiviral activity, etc.). Although such importance of them, their supply still depends principally on the collection of wild plants. Therefore, it is an important issue to develop stable and efficient production system of Glycyrrhiza plants. To overcome this problem, we established the hydroponic cultivation system of Glycyrrhiza uralensis and selected superior G. uralensis clones with high-GL contents in the containment greenhouse. In this study, we aimed to develop a method of selecting these superior G. uralensis clones by DNA sequence polymorphisms in biosynthetic genes. Among the DNA sequences of GL biosynthetic key enzyme gene (CYP88D6), we found Glycyrrhiza species and clone-specific polymorphisms in intronic regions. By using these polymorphisms, discrimination among Glycyrrhiza species and G. uralensis clones became possible. Furthermore, the appearance frequency of superior clone-specific alleles in cloned CYP88D6 sequences was correlated with GL contents in crude drugs collected from the Japanese market. We also observed the tendency that G. uralensis seedlings having superior clone-specific alleles of CYP88D6 gene showed higher secondary metabolite productivity than those without the alleles. These results indicated that superior clone-specific alleles of CYP88D6 gene could be applied as DNA markers for selecting G. uralensis clones accumulating high secondary metabolites.     

Improving the yellow pigment content of bread wheat flour by selecting the three homoeologous copies of Psy1

The yellow pigment content (YPC) of endosperm affects the quality and nutritional value of wheat grain products. Major quantitative trait loci (QTL) for endosperm YPC have been repeatedly mapped on chromosomes 7A and 7B in durum and bread wheats. The genes coding for phytoene synthase (PSY1), which is involved in the biosynthesis of carotenoids, generally co-segregate with these QTL, indicating their role in determining YPC. Here, to study the genetic factors underlying endosperm YPC in bread wheat, the sequence polymorphism of the homoeologous A, B and D copies of genes coding for PSY1, Psy-A1, Psy-B1, and Psy-D1, was studied in a worldwide core collection, which was also phenotyped for flour YPC. Seven novel alleles of Psy-A1 and two novel alleles of Psy-B1 were detected, which confirms the high level of polymorphism of these genes. Two major QTL with respective candidate genes Psy-A1 and Psy-B1 were identified in the distal region of chromosomes 7A and 7B using progeny of a cross between Apache and Ornicar, high and low YPC cultivars, respectively. Association mapping confirms the role of these genes in YPC and shows that the D copy also significantly influences this trait. These results indicate that breeders need to consider all three Psy1 copies when seeking to improve the YPC of wheat endosperm.     

Spatial and developmental expression of key genes of terpene biosynthesis in Tanacetum parthenium

Feverfew (Tanacetum parthenium) is a medicinal plant belonging to the Asteraceae family. To improve understanding terpene metabolism in feverfew, the relative gene expression of four key genes coding 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGR) and germacrene A synthase (GAS) from the mevalonic acid pathway (MVA), as well as 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) from the methyl erythritol phosphate pathway (MEP), were examined. Target organs and tissues included young leaves (not fully expanded), mature leaves (fully expanded), flowers, stems, roots, and glandular trichomes. HMGR, DXR, and HDR were isolated and sequenced for the first time in feverfew. Real-time quantitative PCR analysis revealed differential expression of these genes in feverfew tissues and developmental stages.     

Adaptation of the Osmotolerant Yeast Zygosaccharomyces rouxii to an Osmotic Environment Through Copy Number Amplification of FLO11D

Copy number variations (CNVs) contribute to the adaptation process in two possible ways. First, they may have a direct role, in which a certain number of copies often provide a selective advantage. Second, CNVs can also indirectly contribute to adaptation because a higher copy number increases the so-called “mutational target size.” In this study, we show that the copy number amplification of FLO11D in the osmotolerant yeast Zygosaccharomyces rouxii promotes its further adaptation to a flor-formative environment, such as osmostress static culture conditions. We demonstrate that a gene, which was identified as FLO11D, is responsible for flor formation and that its expression is induced by osmostress under glucose-free conditions, which confer unique characteristics to Z. rouxii, such as osmostress-dependent flor formation. This organism possesses zero to three copies of FLO11D, and it appears likely that the FLO11D copy number increased in a branch of the Z. rouxii tree. The cellular hydrophobicity correlates with the FLO11D copy number, and the strain with a higher copy number of FLO11D exhibits a fitness advantage compared to a reference strain under osmostress static culture conditions. Our data indicate that the FLO gene-related system in Z. rouxii has evolved remarkably to adapt to osmostress environments.     

Mapping of flag smut resistance in common wheat

Flag smut, caused by Urocystis agropyri, has been a problem in wheat production, but its incidence has declined with the use of resistant varieties and seed dressing. Diamondbird, an Australian wheat cultivar that carries high levels of resistance to flag smut, was crossed with susceptible Chinese landrace TH3929 and a doubled haploid (DH) population was developed. A linkage map comprising 386 markers was used for detection of genomic regions controlling flag smut resistance. Composite interval mapping identified five quantitative trait loci (QTL) with significant effects for flag smut resistance. QTL QFs.sun-3AL, QFs.sun-6AS, QFs.sun-1BL and QFs.sun-5BS were contributed by Diamondbird. Although TH3929 was susceptible, it contributed a minor QTL QFs.sun-3AS. QTL QFs.sun-3AL and QFs.sun-6AS were detected in both seasons and each explained more than 17 % of the variation in flag smut response. Other QTL QFs.sun-3AS, QFs.sun-1BL and QFs.sun-5BS explained 5–10 % of the phenotypic variation. DH lines that showed low flag smut levels carried combinations of three or more QTL. This is the first report on chromosomal location of flag smut resistance in a modern common wheat cultivar.     

Validation and application of reference genes for quantitative gene expression analyses in various tissues of <Emphasis Type="Italic">Bupleurum chinense</Emphasis>

It is crucial to select stable references in gene expression analyses using quantitative real-time PCR (qRT-PCR). In this work, seven frequently used reference genes, 18S, Actin, EF1α, α-tubulin, β-tubulin, Cyclophilin and Cytoplasmic ribosomal protein L2 (L2), from Bupleurum chinense DC. were evaluated as the internal control in five tissues, roots, stems, leaves, flowers and fruits, before tissue specific gene expression assays. The results showed that β-tubulin was the most stable and reliable reference gene among the seven candidate genes in the measured tissues. The expression levels of four genes involved in saikosaponins (the pharmacological active compounds of B. chinense) biosynthesis, HMGR, IPPI, FPS and β-AS, were assayed with β-tubulin as the internal control in the five tissues. All the four genes were expressed in the five tissues with different profiles and HMGR in the order of roots > flowers, stems and leaves > fruits, IPPI of stems > leaves and fruits > roots and flowers, FPS of flowers > fruits > stems and roots > leaves and β-AS of roots > flowers, stems and fruits > leaves. The genes of FPS and β-AS were expressed predominantly in flowers and roots, respectively. This study may provide a suitable internal control for quantitative gene expression assays in various tissues and give insight into the tissue expression profiles of four saikosaponins biosynthesis-involved genes of medicinal B. chinense.     

Comparative analyses of squalene synthase (SQS) proteins in poplar and pine by using bioinformatics tools

Squalene synthase (SQS, EC 2.5.1.21) is a major enzyme in biosynthesis of isoprenoid (farnesyl pyrophosphate (FPP) squalene). In the present study, we have analyzed SQS enzymes of black cottonwood (Populus trichocarpa, hereafter Pt) and Masson’s pine (Pinus massoniana, hereafter Pm) using bioinformatics tools. PtSQS and PmSQS sequences were found to have very similar physicochemical properties with “squalene/phytoene synthase” domain structure (PF00494). PtSQS sequence was 47.3 kDa weight and 413 amino acids long with a pI value of 6.86, while PmSQS was 46.6 kDa weight and 409 amino acids long with a pI of 7.92. Alignment of SQS protein sequences in 15 plant species showed a highly similar conserved pattern and included ⁷⁷DTVED⁸¹ and ²¹³DYLED²¹⁷ motifs, which are rich in aspartic acids, for FPP binding sites. In phylogenetic tree, monocots and polycot were clearly separated from dicots with high bootstrap value (99 %). A total of 10 interaction partners were predicted for PtSQS and PmSQS proteins. Nine of them were hypothetical proteins (related with phytosterol biosynthesis), while one was putative uncharacterized protein. Similar 3D structures and identical binding sites were predicted for pine and poplar. In docking, FPP-PtSQS was found to make 8 H bonds with Asp81, Asp217, Glu80, and Gln206 residues in poplar with highest affinity while FPP-PmSQS made 7 H bonds with Arg49, Arg74, Ser48, and Val47 residues in pine with highest affinity. The results of this study will provide valuable theoretical knowledge for future studies of identification and characterization of SQS genes and proteins in various tree species and will provide an insight for studies of biotechnological manipulation of sterol biosynthesis pathway to enhance the plant stress tolerance and productivity.     

Effect of low light intensity on growth and accumulation of secondary metabolites in roots of Glycyrrhiza uralensis Fisch

Effects of low light intensity on growth and accumulation of secondary metabolites of a medicinal plant Glycyrrhiza uralensis Fisch. were investigated. Hydroponic-cultivated one year-old rhizome seedlings were grown under three low irradiances, 200, 100, and 50 μmol m⁻² s⁻¹ for 135 days. Control plants were cultured under natural light conditions. Low light intensity stress decreased leaf thickness, photosynthesis and biomass, but increased leaf area and chlorophyll concentrations. Low light intensity also significantly increased accumulation of glycyrrhizic acid and liquiritin in the root, while the maximum values of both secondary metabolites were obtained under an irradiance of 100 μmol m⁻² s⁻¹. Concentrations of both secondary metabolites were negatively correlated with root biomass. The results suggested that G. uralensis could endure an environment with low light intensity and suitable light control might increase the secondary metabolite contents within agroforestry systems.     

Cloning and characterization of squalene synthase (SQS) gene from Ganoderma lucidum

This report provides the complete nucleotide sequences of the full-length cDNA encoding squalene synthase (SQS) and its genomic DNA sequence from a triterpene-producing fungus, Ganoderma lucidum. The cDNA of the squalene synthase (SQS) (GenBank Accession Number: DQ494674) was found to contain an open reading frame (ORF) of 1,404 bp encoding a 468-amino-acid polypeptide, whereas the SQS genomic DNA sequence (GenBank Accession Number: DQ494675) consisted of 1,984 bp and contained four exons and three introns. Only one gene copy was present in the G lucidum genome. The deduced amino acid sequence of Ganoderma lucidum squalene synthase (Gl-SQS) exhibited a high homology with other fungal squalene synthase genes and contained six conserved domains. A phylogenetic analysis revealed that G. lucidum SQS belonged to the fungi SQS group, and was more closely related to the SQS of U. maydis than to those of other fungi. A gene expression analysis showed that the expression level was relatively low in mycelia incubated for 12 days, increased after 14 to 20 days of incubation, and reached a relatively high level in the mushroom primordia. Functional complementation of Gl-SQS in a SQS-deficient strain of Saccharomyces cerevisiae confirmed that the cloned cDNA encoded a squalene synthase.     

麻疯树鲨烯合酶基因SQS的克隆及生物信息学分析

以麻疯树(Jatropha curcas L.)总RNA为模板,根据已报道的鲨烯合酶基因序列设计简并引物,用RACE方法克隆得到麻疯树鲨烯合酶基因全长cDNA,命名为JcSQS。JcSQS全长1609 bp,包含1个1242 bp的开放阅读框,预测麻疯树鲨烯合酶基因编码的蛋白含有413个氨基酸。JcSQS具有鲨烯合酶类的保守结构域,JcSQS 蛋白与蓖麻、柿、木榄等植物中SQS基因编码的氨基酸序列具有高度同源性。这为研究麻疯树萜烯类物质的生物合成和调控机制奠定了基础。